The present invention relates to compositions and methods for diagnosing and/or detecting and/or preventing and/or treating Candida albicans or conditions or symptoms associated therewith, as well as to process and products for preparing such compositions and methods.
The present invention further relates to CaESS1, an important Candida albicans gene, e.g., nucleic acid molecules therefor, and/or fragments or portions thereof, expression products therefrom, e.g., the protein CaEss1 or fragments or portions thereof, methods for making and using the gene, portions thereof and expression products therefrom, and to targeting the gene or portions thereof and/or the expression products therefrom for antifungal applications.
The identification of the CaESS1 gene allows for identifying compounds or agents that specifically bind to and/or inhibit the gene, or portions thereof and/or expression products therefrom, and methods for preventing and/or treating Candida albicans and/or symptoms or conditions associated therewith, as well as generally for making and using such compounds or agents. Thus, the invention relates to antifungal preparations and/or compositions and methods for making and using them.
The CaEss1 amino acid sequence and the CaESS1 DNA or nucleic acid sequences can be used for diagnostic purposes. For instance, the nucleic acid sequences can be used to generate primers for diagnostic DNA, and the invention comprehends such primers. Primers are preferably derived from those parts of the CaESS1 gene which are least conserved among the ESS1/PIN1 family members. The gene or the primers can be used to detect if the gene is present in a sample or specimen and/or if the gene was expressed as RNA in a sample or specimen. Accordingly, the invention relates to compositions and methods for detecting and/or diagnosing Candida albicans. 
The CaESS1 gene and portions thereof are useful for generating or expressing the CaEss1 protein and epitopic portions thereof (epitopic portions of the protein can be derived from, generated by, or expressed from those parts of the CaESS1 gene which are least conserved among the ESS1/PIN1 family members). The protein or portions thereof is useful for generating antibodies, such as monoclonal and/or polyclonal antibodies. These antibodies can be used for diagnostic purposes; and, the protein or portions thereof can be used for diagnostic purposes, e.g., the antibodies can be used to detect or determine, e.g., via binding, whether the proteins or portions thereof are present in a sample or specimen and the protein or portions thereof can be used to detect or determine, e.g., via binding, whether antibodies thereto are present in a sample or specimen. Further, the antibodies can be used to block CaEss1 activity. Accordingly, the invention relates to diagnostic compositions and methods, as well as therapeutic or preventive compositions and methods.
The invention further relates to methods for screening compounds for the ability to inhibit CaEss1 and/or PIN1. Compounds which selectively inhibit CaEss1 and do not inhibit PIN1 or do not inhibit PIN1 greatly are compounds useful in the prevention and/or treatment of Candida albicans. Compounds which inhibit PIN1 are useful in antiproliferative applications, e.g., as antineoplastic, anti-tumor or anticancer agents. Furthermore, the screening methods of the invention can be adapted and used for screening compounds which inhibit other fungal infection as fungus other than Candida albicans have ESS1 genes. Accordingly, the invention relates to methods for screening for inhibitors of CaEss1, PIN1 or other ESS1s, as well as to inhibitors of these enzymes.
Various documents are cited in this text, or in a reference section preceding the claims. Each of the documents cited herein, and each of the documents cited or referenced in each of those various documents, is hereby incorporated herein by reference. None of the documents cited in the following text is admitted to be prior art with respect to the present invention.
Candida albicans is an asexual yeast species. Candida albicans is a major fungal pathogen of humans. It is can be found as a harmless commensal organism, inhabiting mucosal membranes and the digestive tract; a benign saprophyte. However, Candida albicans, can infect both internal organs and mucous membranes of the mouth, throat, and genital tract, and can cause a chronic infection; it can cause superficial infections, such as oral thrush, and can cause severe, often fatal, systemic infections, especially in immunocompromised patients.
There has been a growing number of cases of thrush and other diseases caused by Candida albicans; and, this can be attributed mainly to medical advances in antibiotic, steroid and immunosuppressive treatments, as well as to immunocompromising ailments such as HIV and AIDS. Indeed, surveillance of nosocomial blood stream infections (BSI) in the USA between April 1995 and June 1996 revealed that Candida albicans was the fourth leading cause of nosocomial BSI (Pfaller et al., xe2x80x9cNational surveillance of nosocomial blood stream infection due to Candida albicans: frequency of occurrence and antifungal susceptibility in the SCOPE Program,xe2x80x9d Diagn Microbiol Infect Dis 1998 May;3 1(1):327-32). Accordingly, Candida albicans, and compositions and methods for detecting, diagnosing, preventing or treating Candida albicans are medically significant.
Thrush is characterized by creamy-white, curdlike patches on the tongue and other mucosal surfaces of the mouth. The disease is caused by an overgrowth of Candida albicans. Patients susceptible to thrush include immunocompromised individuals, e.g., adults whose immune systems have been weakened by antibiotics, steroids, immunosuppression treatments, AIDS, and the like, as well as infants, for instance if the mother had a vaginal yeast infection.
Painful, raw and bleeding areas result if the curdlike discharge is removed from patches of thrush. These superficial lesions may allow the yeast to spread to other areas of the body. Candida albicans can invade major organs, causing serious complications.
While thrush is typically treated with a topical agent, and there are oral and intravenous treatments for Candida albicans infections, chronically infected patients may require long term therapy with oral and/or intravenous therapy.
Moreover, strains of Candida albicans resistant to present treatments or therapies such as amphotericin B, fluconazole, itraconazole and other azole antifungals have been isolated (Mori et al., xe2x80x9cAnalysis by pulsed-field gel electrophoresis of Candida albicans that developed resistance during antifungal therapy,xe2x80x9d Nippon Ishitkin Gakkiai Zasshi 1998;39(4):229-33; Pfaller et al., supra; Rex et al., xe2x80x9cA randomized trial comparing fluconazole with amphotericin B for the treatment of candidemia in patients without neutropenia,xe2x80x9d N Engl J Med 1994 Nov 17;331(20):1325-30). Indeed, in Rex et al., in certain individuals, treatment failed to clear infection from the bloodstream, and Candida albicans was infection commonly associated with the treatment failure.
Thus, there is a need for new treatments or therapies against Candida albicans. 
Diagnosis of Candida albicans requires microscopic identification of the pseudomycelial (branching-arms) forms. There is likewise a need for new compositions and methods for diagnosing or detecting Candida albicans. 
The ESS1 gene was originally discovered in Saccharomyces cerevisiae, by inventor Hanes working in the laboratory of Dr. Peter Shank and Dr. Keith Bostian (Hanes 1988). It was discovered in a search for cell growth control genes. By gene disruption techniques, ESS1 was shown to be essential for yeast cell growth, hence the name (Essential) (Hanes et al. 1989). ESS1 genes are highly conserved. Homologs of the ESS1 gene have been found in Drosophila, humans and several other organisms. The fly gene (called dodo) and the human gene (called PIN1) encode proteins that are 45% identical to the yeast Ess1 protein (Maleszka et al. 1996; Lu et al. 1996).
WO 97/17986 relates to the identification and characterization of Pin1, a protein of mammalian origin that associates with NIMA protein kinase. It was determined that overexpression of Pin1 activity induces a specific G2 arrest and delays NIMA-induced mitosis, while depletion of Pin1 triggers mitotic arrest and nuclear fragmentation. The specification provides for a method of controlling the growth of a cell by contacting the cell with a composition which modulates the Pin1 activity. See also U.S. Pat. Nos. 5,952,467 and 5,972,697.
However, prior to the present invention, the Candida albicans ESS1 or CaESS1 gene had not been isolated, or sequenced; or disclosed or suggested, nor had corresponding amino acid sequences from the gene been disclosed or suggested. Likewise, fragments or portions of the gene and protein had not been disclosed or suggested. Also, diagnostic, prophylactic, therapeutic, or similar compositions and methods involving the gene and/or the protein and/or fragments of the gene and/or fragments of the protein, had not been taught or suggested.
In view of the significance of Candida albicans, and the need for new therapies, treatments, means for prevention, and means for diagnosing or detecting Candida albicans, providing the CaESS1 gene, portions thereof, amino acid sequences from the gene, fragments or portions of the protein, and diagnostic, prophylactic, therapeutic, or similar compositions and methods involving the gene and/or the protein and/or fragments of the gene and/or fragments of the protein, are significant advances in the art, addressing problems in the art.
An object of the invention can include providing any or all of: the CaESS1 gene, portions thereof, amino acid sequences from the gene, fragments or portions of the protein, and diagnostic, prophylactic, therapeutic, or similar compositions and methods involving the gene and/or the protein and/or fragments of the gene and/or fragments of the protein.
Accordingly, the present invention provides an isolated and/or purified nucleic acid molecule encoding CaEss1, e.g., CaESS1; for instance, an isolated and/or purified nucleic acid molecule comprising a nucleotide sequence encoding CaEss1 as set forth in FIG. 1 (SEQ ID NO: 1).
The present invention also provides an isolated and/or purified nucleic acid molecule which is a primer for an isolated and/or purified nucleic acid molecule encoding CaEss1, e.g., CaESS1, for instance, a primer for an isolated and/or purified nucleic acid molecule comprising a nucleotide sequence encoding CaEss1 as set forth in FIG. 1. Such a primer can be OW-216 or OW-221 disclosed below (SEQ ID NOS: 3, 6).
The present invention also provides an isolated and/or purified CaEss1 protein; for instance, such a protein from expression of any or all of the foregoing nucleic acid molecules, or as shown in FIG. 1 (SEQ ID NO:2).
The invention further provides nucleic acid molecules and amino acid molecules having at least 70%, e.g., at least 75%, such as at least 80%, e.g., at least 85%, preferably at least 90%, more preferably at least 95% such as at least 97% homology, identity or similarity to such molecules disclosed herein.
The invention further provides diagnostic compositions and methods involving the nucleic acid molecules, as well as the amino acid molecules or antibodies generated therefrom.
Thus, the invention further provides methods for determining the presence of Candida albicans in a sample; for instance, by detecting the presence in the sample of CaESS1 e.g., by diagnostic PCR using a primer or probe specific for CaESS1; or, by detecting CaEss1 by contacting the sample with an antibody specific for CaEss1 and detecting binding of the antibody; or by detecting antibodies to CaEss1 by contacting the sample with an inventive amino acid molecule and detecting binding thereof to an antibody in the sample.
The invention further provides therapeutic or preventive compositions, e.g., compositions useful for treating or preventing a fungal infection such as a Candida albicans infection or for antiproliferative effect, e.g., antineoplastic, anti-tumor or anti-cancer effect, as well as to methods for treating or preventing such fungal infections or cell proliferation.
A CaEss1 inhibitor can be a compound which selectively inhibits growth of S. cerevisiae not containing an endogenous ESS1 gene but rather CaESS1 and uninduced PIN1 (e.g., on a glucose medium, see FIGS. 2, 3) and/or preferably does not inhibit or significantly inhibit induced PIN1, e.g., does not inhibit or significantly inhibit S. cerevisiae not containing an endogenous ESS1 gene but rather induced PIN1 (see FIG. 3). An anti-CaEss1 antibody or an antibody against an epitopic region of CaEss1 can also be an inhibitor of CaEss1 by virtue of the antibody being able to bind to CaEss1. Compositions which indeed inhibit PIN1 are nonetheless useful as antiproliferatives, e.g., antineoplastics, anti-tumor agents or anti-cancer agents.
The invention comprehends methods for preventing or treating Candida albicans or cancer by administering the inventive compositions.
Further still, the invention provides methods for screening compounds for inhibiting Candida albicans, as well as other fungal infectious agents, and human cell growth. The screening method entails plating yeast transformed to express a fungal ESS1 gene such as CaESS1 alone or with PIN1, and contacting those yeast with a potential inhibitory compound, whereby compounds specifically inhibit yeast cell growth when CaESS1 is expressed, but not when PIN1 is expressed. Such compounds are specific inhibitors of ESS1 such as CaEss1, but not PIN1 .
The invention yet further still entails a method for screening for antiproliferative compounds, e.g., anti-tumor, anticancer or antineoplastic agents, comprising plating yeast transformed to express low or high levels of PIN1 (e.g., on glucose/galactose and glucose media) and determining compounds which selectively inhibit growth of yeast expressing low levels of PIN1, e.g., on the glucose/galactose medium, but not growth of yeast expressing high levels of PIN1, e.g., on the galactose medium.
The terms xe2x80x9ccomprises,xe2x80x9d xe2x80x9ccomprising,xe2x80x9d and the like can have the meaning ascribed to these terms in U.S. Patent Law and can mean xe2x80x9cincludes,xe2x80x9d xe2x80x9cincluding,xe2x80x9d and the like.
These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.